Signalling by the two second messengers, cAMP and Ca2+, is central to the control of numerous physiological processes by hormones and neurotransmitters. The discovery of Ca2+-sensitive (stimulable and inhibitable) adenylyl cyclases in heart, discrete brain regions,pituitary, platelets, etc. provides a viable means whereby these two signalling systems could be tightly coordinated. This potentially important role for Ca2+- sensitive adenylyl cyclases rests on whether physiological elevation of cytosolic Ca2+ ([Ca2+]) causes the changes in activity that are anticipated from in vitro studies. A guiding premise in the present project is the need to understand clearly how [Ca2+]i is distributed both within cellular domains and over time. This foundation greatly enriches the questions asked and the quality of the information derived. Current research from this lab indicates that indeed physiological [Ca2+]i-rises inhibit the endogenous Ca2+-inhibitable adenylyl cyclase in cell lines such as C6-2B gliomas. However, very strikingly, when the inhibitory efficacy of the entry and release component of a rise in [Ca2+]i are separately evaluated, only Ca2+-entry inhibits cAMP synthesis, while release is without effect. In addition, in HEK 293 cells, transfected with various adenylyl cyclase cDNAs, Ca2+-entry is sufficient to regulate both Ca2+- inhibitable and -stimulable adenylyl cyclases. Therefore, it is now proposed to ask whether this dependence on entry is a predetermined outcome for a cyclase that is expressed in C6-2B cells, because Ca2+-entry in C6=2B cells is particularly efficacious in delivering [Ca2+]i to the cyclase, or whether this sensitivity is a property intrinsic to Ca2+-sensitive adenylyl cyclases, even when they are transfected in HEK 293 cells. These issues will be addressed by imaging experiments in single C6-B cells, to determine whether different concentrations of Ca2+ are achieved at the plasma membrane in response to Ca2+ -entry or -release, elicited by various means; parallel immunohistochemical experiments will also be performed to ask whether adenylyl cyclases are found in regions of high Ca2+ (so-called caveolae) in C6-2B cells. Whether the same dependence on entry vs. release is displayed by adenylyl cyclase cDNAs expressed in HEK 293 cells will be determined, as a prelude to exploring whether the in vivo sensitivity to Ca2 can be separated from the in vitro sensitivity by molecular biological manipulation of cyclase cDNAs, using chimeric and deletion approaches. These studies will be complemented by experiments that probe the domains of cyclases that are responsible for their in vitro sensitivity to Ca2+. This project then combines cell biological and molecular studies of [Ca2+]i and cAMP in an attempt to establish the likely critical roles played by Ca2+ - sensitive adenylyl cyclases in cellular signalling.